Bacterial strain, processed plant extracts, compositions containing same, processes for their preparation and their therapeutic and industrial applications

ABSTRACT

The present invention discloses: (i) a non-pathogenic probiotic microorganism and its probiotic/therapeutic uses; (ii) a formulation comprising an aqueous solution of a volatile fraction (VF) prepared from the extract of at least one plant derived material and its therapeutic uses; (iii) a process of manufacturing the formulation from the plant derived material; (iv) a probiotic composition comprising the non-pathogenic probiotic microorganism of the invention and/or other probiotic microorganism(s) and the formulation of the invention, and its probiotic/therapeutic uses; (v) a composition for industrial applications comprising the formulation of the invention and microorganism(s) of industrial applicability; and (vi) industrial processes and apparatuses in which the latter composition is used.

[0001] This is a continuation-in-part of PCT Application No. IL00/00318,filed Jun. 1, 2000.

FIELD AND BACKGROUND OF THE INVENTION

[0002] The present invention relates to (i) a non-pathogenic probioticmicroorganism and its probiotic/therapeutic uses; (ii) a formulationcomprising an aqueous solution of a volatile fraction (VF) prepared fromthe extract of at least one plant derived material and its therapeuticuses; (iii) a process of manufacturing the formulation from the plantderived material; (iv) a probiotic composition comprising thenon-pathogenic probiotic microorganism of the invention and/or otherprobiotic microorganism(s) and the formulation of the invention, and itsprobiotic/therapeutic uses; (v) a composition for industrialapplications comprising the formulation of the invention andmicroorganism(s) of industrial applicability; and (vi) industrialprocesses and apparatuses in which the latter composition is used.

[0003] Under normal conditions, the gastrointestinal (GI) tractmicroflora contributes significantly to the health and well being of anindividual. It is well known that the microflora is a complex anddiverse population of microorganisms, which may have both beneficial andharmful effects on the individual. Under normal gestation conditions,the fetus in utero is sterile, but on passage through the vagina duringbirth it acquires microorganisms resulting in the formation of a gutmicroflora. The final indigenous gut microflora which stabilizes in thegut is a very complex collection of over one thousand different types ofmicroorganisms, consisting of about 400 different types of bacteria[Fuller R. J. Applied Bacteriology 66:365-378 (1989)]. The compositionof the gut microflora is determined by both host and microbial factors,and although there are a lot of bacteria which can survive and grow inthe GI tract, there are many which cannot. In addition, the survivingorganisms have to avoid the effect of peristalsis which normally flushesout the bacteria with the food. This may be achieved by the bacteriaimmobilizing themselves by attachment to the gut wall, and/or bymultiplying at a rate that exceeds the rate of removal by peristalsis.

[0004] In general, the presence of gut microflora is symbiotic as themicroflora not only assists in disintegrating some undigestable foods,it also protects the individual from infections caused by pathogens.This latter phenomenon has been described under such names as ‘bacterialantagonism’, ‘bacterial interference’, ‘barrier effect’, ‘colonizationresistance’, ‘competitive exclusion’ and many others.

[0005] The gut protective microflora is very stable. However, it is lesseffective in the young, elderly and the compromised patient. Further, itcan be influenced by certain dietary and environmental factors, thethree most important being excessive hygiene, antibiotic therapy andstress.

[0006] Under conditions where the balance of the gut microflora isadversely affected, probiotics become of potential value in restoringthe gut microflora and enabling the individual host to return to normal.

[0007] Probiotics are a class of microorganisms defined as livemicrobial organisms that beneficially affect the animal and human hosts.The beneficial effects include improvement of the microbial balance ofthe intestinal microflora or improving the properties of the indigenousmicroflora. The beneficial effects of probiotics may be mediated by adirect antagonistic effect against specific groups of organisms,resulting in a decrease in numbers, by an effect on their metabolism orby stimulation of immunity. Probiotics may suppress viable counts of anundesired organism by producing antibacterial compounds, by competingfor nutrients or for adhesion sites. Further, they may alter microbialmetabolism by increasing or decreasing enzyme activity or they maystimulate the immune system by increasing antibody levels or increasingmacrophage activity.

[0008] WO95/16461 describes a probiotic composition of anaerobicbacteria effective in controlling or inhibiting Salmonella colonizationin domesticated animals. The probiotic composition includes populationsor cultures of 29 substantially biologically pure bacteria, inter alia,E. coli. However, the suppression of the pathogen by the probioticcomposition described in this PCT publication requires the combinedaction of a large number of bacterial strains.

[0009] WO97/35596 describes the administration of a freshly preparedprobiotic mixture obtained by mixing a powder containing Lactobacillusreuteri, Lactobacillus acidophilus and Bifidobacterium infantis with aliquid. The mixture is described to be effective in preventinginfectious diarrhea or diarrhea caused by antibiotic therapy in humans.The freeze-dried live bacteria are, however, in anabiotic state. Theneed to wet the microorganism before administration, in order toreinstate its vitality, is a disadvantage, since normally many bacteriado not survive the re-hydration. Moreover, the surviving organisms arenot immediately metabolically active, and cannot survive the extreme,acidic conditions of the stomach. Furthermore, when administered to arecipient with diarrhea, the rate of their removal from the gut mayexceed the rate of reinstation of viability, resulting in minimal or nobeneficiary effect.

[0010] Preservation of viability and conservation of the activity ofprobiotic organisms by their formulation is the issue of numerouspublications. WO98/26787 describes the enhancement of a residentpopulation of lactic acid-producing microorganisms, preferablylactobaccillii, in the GI tract of an animal by providing the same withβ-glucan, optionally in combination with prebiotic and/or probioticmicroorganisms.

[0011] WO97/34591 also describes the enhancement of resident populationof microorganisms, or the suppression of the undesired residentpopulation at a selected site of the GI tract of an individual, byproviding the individual with a selected modified or unmodified starchor mixtures thereof, which act as carrier for one or more probioticmicroorganisms and as a growth or maintenance medium for themicroorganisms. The probiotic elements are bound to the carrier in amanner so as to protect the microorganisms during passage to the largebowel or other regions of the GI tract.

[0012] Microorganisms are used in a variety of industrial processes.These processes can be divided into continuous processes and batchprocesses. In continuous processes, such as a continuous aerobic oranaerobic fermentation line, used for the production of a variety offermentation products, including, for example, ethanol and methanol, afermentable raw material is continuously fed into the line and thefermentive product is continuously collected from the line, in acontrolled fashion, such that the microorganism population in the lineis sustained throughout the process. In batch processes, such as, butnot limited to, batch aerobic or anaerobic fermentation, biodegradationof oil in, for example, oil spills, and the like, a fermentable rawmaterial is mixed with a population of microorganism(s) so as to instatefermentation. Both in continuous fermentation and in batch fermentationprocesses, it is desired, in some cases, to start or reinstate theprocess with a large and viable population of the relevantmicroorganism(s). For example, when biodegrading oil, it is advantageousto start the process with an as large as possible population of themicroorganisms Pseudomonas spp. or Alcaligenes spp., for example, so asto minimize the time for complete biodegradation of the oil. When usingfilters enriched with microorganism(s) for biodegradation of organicfumes or volatiles, the result is a continuous process in whichpolluting compounds are degraded into harmless compounds. However, themicroorganisms present in the filter depend for their survival oncontinuous supply of organic fumes or volatiles. Nevertheless, in someinstances the supply of organic fumes or volatiles is discontinued and,as a result, the microorganism(s) are lost. Under such circumstances,reinstation of an effective viable amount of microorganism(s) in thefilter is required in a short period of time, so as permit immediaterestoration of a functional filter.

[0013] There is thus a widely recognized need for, and it would behighly advantageous to have, a formulation in which microorganisms,including probiotic microorganisms and microorganisms useful in avariety of industrial applications, could be maintained viable and in ametabolically active form for long periods of time.

[0014] While reducing the present invention to practice, it has beenunexpectedly found that a single species of a non-pathogenic probioticmicroorganism derived from E. coli is, alone, capable of restoringnormal GI flora of man and of a variety of mammals and avians. It hasalso been surprisingly found that this microorganism, as well as othermicroorganisms, could be preserved for long periods of time, in a viableand metabolically active form, in a formulation comprising watersolution of volatile fraction(s) of various plant extracts. A probioticcomposition comprising the probiotic organism suspended in theformulation was found to be effective in the treatment and prevention ofvarious gastrointestinal disorders. It has further been unexpectedlyfound that the formulation per se is effective as a body weight gainenhancer and as an immuno-stimulator in mammals and avians.

SUMMARY OF THE INVENTION

[0015] According to one aspect of the present invention there isprovided an Escherichia coli strain BU-230-98 ATCC Deposit No. 20226(DSM 12799).

[0016] According to another aspect of the present invention there isprovided a probiotic composition comprising viable Escherichia colistrain BU-230-98 ATCC Deposit No. 20226 (DSM 12799) and a formulationfor maintaining viability of the Escherichia coli strain. The probioticcomposition preferably further comprising at least one flavouring agent.Preferably, the formulation includes at least one volatile fraction (VF)of a plant extract prepared by steam distillation of a plant (e.g.,beet, dill, parsley or grapefruit) extract under reduced pressure and ata bath temperature preferably not exceeding 38° C.

[0017] The probiotic composition of the present invention may beidentified for preventing or treating gastro-enteric infections ordisorders, maintaining or reinstating normal gastro-intestinalmicroflora, preventing or treating diarrhea, preventing or treatinggastro-enteric infection caused by an enteric pathogen, such as a Gramnegative bacterium or Gram positive bacterium, preventing or treatinggastro-enteric Salmonella infection, preventing or treating infectiousdiarrhea, caused by, for example C. difficile, Salmonella, particularlyS. Shigella, Campylobacter, E. coli, Proteus, Pseudomonas orClostridium, chronic diarrhea or diarrhea resulting from antibiotictherapy, radiotherapy or chemotherapy, and/or for normalizing thephysiological activity of the gastrointestinal tract.

[0018] According to yet another aspect of the present invention there isprovided a formulation comprising at least one volatile fraction (VF) ofa plant extract, the volatile fraction is prepared by steam distillationof the plant extract under reduced pressure and at a bath temperaturenot exceeding 38° C. The plant extract is preferably obtainable from aplant organ selected from the group consisting of leaves, stems, rootsand fruit. The plant can be a vegetable, such as soy bean, alfalfa,garlic, beet and cabbage, or a herb, such as parsley, mint and dill. Theformulation may further include a beehive product, such as, but notlimited to, propolis. The formulation can serve as a veterinary feed orfood additive for enhancing animal weight gain in a recipient. It canalso serve to preserve viable bacteria as is further described herein.

[0019] Hence, according to an additional aspect of the present inventionthere is provided a process of preparing a volatile fraction of a plant,comprising the steps of (a) grinding a plant derived material to give aplant biomass; (b) mixing the plant biomass with water and stirring atambient temperature; (c) steam distilling the mixture obtained in step(b) under reduced pressure and at a bath temperature not exceeding 38°C.; and (d) collecting volatile fraction obtained from the steamdistillation. Preferably, the reduced pressure is of 5-10 mbar. Thisformulation can be used as a food additive, a feed additive and hasunexpected therapeutic uses, as well as advantageous bacteriapreservation properties. A therapeutic formulation containing thevolatile fraction of a plant is also disclosed and may be prepared bymixing at least one volatile fraction (VF) of a plant extract withphysiologically or veterinary acceptable additives, carriers ordiluents. Preferably, the volatile fraction is prepared by steamdistillation of the plant extract under reduced pressure and at a bathtemperature not exceeding 38° C.

[0020] Thus, according to still another aspect of the present inventionthere is provided a probiotic composition comprising (a) an effectiveamount of at least one viable probiotic microorganism having abeneficial biological or therapeutic activity in the gastrointestinaltract; and (b) at least one volatile fraction (VF) of a plant extractpreferably prepared as described herein. The probiotic microorganism canbe E. coli, preferably, strain BU-230-98, ATCC Deposit No. 202226 and itcan be identified for treatment or prevention of any of the abovedisorders/pathologies/diseases/syndromes and in addition or as analternative in can be identified for treatment or prevention ofdyspeptic symptoms, for enhancing the immune response in a patientsuffering from an immune disorder, resulting from, for example,immune-response suppression therapy.

[0021] According to still another aspect of the present invention thereis provided a composition comprising (a) at least one microorganism,such as, Escherichia spp., Alcaligenes spp., Arthrobacter spp.,Bifidobacterium spp., Lactobacillus spp., Lactococcus spp., Nitrosomonasspp. and Pseudomonas spp.; and (b) at least one volatile fraction (VF)of a plant extract, the at least one volatile fraction is selected so asto sustain viability of the at least one microorganism for at least 2months, preferably at least 3-6 months, more preferably 6-12 months ormore at room temperature.

[0022] According to another aspect of the present invention there isprovided a process of growing Escherichia coli strain BU-230-98 ATCCDeposit No. 20226 (DSM 12799), the process comprising the step ofseeding a starter cloture of Escherichia coli strain BU-230-98 ATCCDeposit No. 20226 (DSM 12799) into a growth media and growing theEscherichia coli strain up to optical density of 15-30 at 650 nm.

[0023] According to yet another aspect of the present invention there isprovided a process of manufacturing a probiotic composition, the processcomprising the step of suspending viable Escherichia coli strainBU-230-98 ATCC Deposit No. 20226 (DSM 12799) in a formulation formaintaining viability of the Escherichia coli strain. Preferably, theformulation includes at least one volatile fraction (VF) of a plantextract, prepared, preferably by steam distillation of the plant extractunder reduced pressure and at a bath temperature not exceeding 38° C.,obtained from, for example, a plant organ selected from the groupconsisting of leaves, stems, roots and fruit, either a vegetable and aherb, such as soy bean, parsley, mint, dill, alfalfa, garlic, beet orcabbage.

[0024] According to still another aspect of the present invention thereis provided a process of preparing a probiotic composition, the processcomprising the step of suspending an effective amount of at least oneviable probiotic microorganism having a beneficial biological ortherapeutic activity in the gastrointestinal tract in an formulationcontaining at least one volatile fraction (VF) of a plant extract.

[0025] According to an additional aspect of the present invention thereis provided a process of preparing a composition including viablemicroorganisms, the process comprising the step of suspending at leastone microorganism in a formulation containing at least one volatilefraction (VF) of a plant extract, the at least one volatile fraction isselected so as to sustain viability of the at least one microorganismfor at least 2 months at room temperature. The microorganism can be, forexample, Escherichia spp., Alcaligenes spp., Arthrobacter spp.,Bifidobacterium spp., Lactobacillus spp., Lactococcus spp., Nitrosomonasspp. or Pseudomonas spp.

[0026] According to yet an additional aspect of the present inventionthere is provided a dispenser of microorganisms comprising a reservoirand a dispensing mechanism being connected thereto, the reservoirhousing at least one microorganism in a formulation containing at leastone volatile fraction (VF) of a plant extract, the at least one volatilefraction is selected so as to sustain viability of the at least onemicroorganism for at least 2 months at room temperature.

[0027] According to still an additional aspect of the present inventionthere is provided a method of biocontrol of an organism, the methodcomprising the step of spreading in an endemic area at least one viablemicroorganism capable of biocontrolling the organism, the at least oneviable microorganism being in a formulation containing at least onevolatile fraction (VF) of a plant extract, the at least one volatilefraction is selected so as to sustain viability of the at least onemicroorganism for at least 2 months at room temperature.

[0028] According to a further aspect of the present invention there isprovided a method of eliminating an oil pollution, the method comprisingthe step of spreading in a polluted area at least one viablemicroorganism capable of degrading oil, the at least one viablemicroorganism being in a formulation containing at least one volatilefraction (VF) of a plant extract, the at least one volatile fraction isselected so as to sustain viability of the at least one microorganismfor at least 2 months at room temperature.

[0029] According to yet a further aspect of the present invention thereis provided a method of restoring organic fumes degrading microorganismpopulation in a biofilter, the method comprising the step of dispensingonto the filter at least one viable microorganism capable of degradingorganic fumes, the at least one viable microorganism being in aformulation containing at least one volatile fraction (VF) of a plantextract, the at least one volatile fraction is selected so as to sustainviability of the at least one microorganism for at least 2 months atroom temperature.

[0030] According to still a further aspect of the present inventionthere is provided a method of preparing a starter for a fermentationprocess, the method comprising the step of growing a sufficient amountof a starter microorganism and suspending the starter microorganism in aformulation containing at least one volatile fraction (VF) of a plantextract, the at least one volatile fraction is selected so as to sustainviability of the starter microorganism for at least 2 months at roomtemperature.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

[0032] In the drawings:

[0033]FIG. 1 is a schematic depiction of a dispenser according to thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention is of (i) a non-pathogenic probioticmicroorganism and its probiotic/therapeutic uses; (ii) a formulationcomprising an aqueous solution of a volatile fraction (VF) prepared fromthe extract of at least one plant derived material and its therapeuticuses; (iii) a process of manufacturing the formulation from the plantderived material; (iv) a probiotic composition comprising thenon-pathogenic probiotic microorganism of the invention and/or otherprobiotic microorganism(s) and the formulation of the invention, and itsprobiotic/therapeutic uses; (v) a composition for industrialapplications comprising the formulation of the invention andmicroorganism(s) of industrial applicability; and (vi) industrialprocesses and apparatuses in which the latter composition is used.

[0035] The principles and operation of the present invention may bebetter understood with reference to the drawings and accompanyingdescriptions.

[0036] Before explaining at least one embodiment of the invention indetail, it is to be understood that the invention is not limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways. Also,it is to be understood that the phraseology and terminology employedherein is for the purpose of description and should not be regarded aslimiting.

[0037] In one aspect, the present invention relates to a probioticmicroorganism being a non-pathogenic bacterium derived from E. coli,having a beneficial physiological and/or therapeutic activity in, forexample, the GI tract and deposited at the ATCC under deposit No. 202226and at DSMZ under deposit No. 12799.

[0038] “Probiotic” is used herein as an adjective to describe anisolated bacteria having the property of inhibiting the growth of atleast one pathogen. The test of an inhibition used herein was an invitro test on solid medium in which culture supernatants of candidateisolated bacteria were observed for their property of inhibiting thegrowth of a pathogen when applied to the surface of the solid medium.Typically, a paper disc impregnated with the culture supernatant of acandidate probiotic strain was placed on the surface of an agar plateseeded with the pathogen. Probiotic bacterial supernatants caused a ringof clear agar or of reduced growth density indicating inhibition of thepathogen in the vicinity of the disc. There are other tests forinhibition which are available or could be devised, including directgrowth competition tests, in vitro or in vivo which can generate a panelof probiotic bacteria similar to that described herein. The bacterialstrains identified by any such test are within the category of probioticbacteria, as the term is used herein.

[0039] The probiotic bacteria of the present invention may serve as afood or feed additive, so as to provide food or feed productsupplemented with viable Escherichia coli strain BU-230-98 ATCC DepositNo. 20226 (DSM 12799). A food product of the present invention mayfurther include at least one milk derived substance and may be selectedfrom the group consisting of a cheese and a yogurt.

[0040] The present invention further relates to a formulation comprisingat least one volatile fraction (VF) of a plant extract, the volatilefraction being prepared by distillation of the plant extract underreduced pressure and at a bath temperature typically not exceeding 38°C.

[0041] The plant matter from which the volatile fraction may be obtainedmay be any suitable plant part such as fruit, leaf, stem or root. Manyplants are suitable as a source for the volatile fractions, for exampleapple, citrus, soy bean, beet, cabbage, garlic and alfalfa, as well asherbs such as parsley, mint and dill. The formulations of the inventionmay optionally further comprise a suitable amount of a volatile fractionof an apicultural product such as honey, propolis or other beehiveproduct, which may be prepared in the same manner as described hereinfor obtaining volatile fractions from plant extracts.

[0042] As will be described in more detail in the following Examples,the formulation itself may be used as a food/feed additive. It has beenfound by the inventors to have a weight gain enhancing activity and itconstitutes an aspect of the invention. According to a particularembodiment of this aspect of the invention, the animal weight gainenhancing formulation or feed additive comprises distilled water andvolatile fractions of alfalfa, soy beans, beet and dill, preferably at avolume ratio of the volatile fractions of 2:8:1:4. Alternatively, aformulation or feed additive of the present invention comprisesdistilled water and volatile fractions of carrot, beet, dill andgrapefruit (1 part each) per 100 parts of water. Still alternatively, aformulation or food additive of the present invention comprisesdistilled water and volatile fractions of beet, dill and parsley (1 parteach) per 100 parts of water.

[0043] In a further aspect, the invention relates to a compositioncomprising the above formulation of the invention and at least oneviable microorganism, either a probiotic organism having a therapeuticor beneficial biological activity in, for example, the GI tract of humanand/or animal, to thereby provide a probiotic composition, or anorganism having industrial applications, to thereby provide acomposition of industrial applicability.

[0044] A particular advantage of the probiotic and industrialcompositions of the invention stems from the fact that they are liquidpreparations. Being under biologically active conditions, theformulation serves also as a supportive medium for living bacteria, asopposed to lyophilized formulations where the bacteria are in ananabiotic condition. As a result, the probiotic composition of theinvention, for example, is active immediately following oraladministration, beginning with the upper portion of the GI tract, whereprimary effects of the majority of intestinal pathogens take place,causing development of adverse gastro-enteric syndromes. The probioticcompositions of the invention may also be used as body weight-increasingpreparations or food/feed additives. Similarly, the industrialcomposition of the invention can be used to efficiently reinstate apopulation of microorganisms of industrial applicability.

[0045] Thus, it is noted that, for probiotic applications, probioticbacteria other than such belonging to the strain BU-230-98 (ATCC DepositNo. 202226, also deposited at the DSM under No. 12799), may be used withthe plant material derived formulation of the invention. Such probioticbacteria have a very broad spectrum of antagonistic activity. They alsobelong to the same phylogenetic group of the majority of intestinalpathogens and share the same systems of survival. Therefore, thesuppression and exclusion of intestinal pathogens may include manydifferent mechanisms, for example, secretion of antagonistic material,competition for utilization of nutrients and competition for adhesionreceptors. Thus, any non-pathogenic bacteria which comply with thesecriteria, may be used in the probiotic compositions of the invention.

[0046] According to a further aspect of the invention, the probioticcompositions of the invention may be used for preventing or treatinggastro-enteric infections. Term ‘gastro-enteric infection’ is to betaken to mean any infection caused an enteric pathogen, including, interalia, Gram negative and Gram positive bacteria. By improving the generalbalance and health of the GI tract, the formulations and probioticcompositions of the invention may be instrumental in prophylaxis of alsoGI infections caused by yeast, viruses and protozoa.

[0047] The term ‘therapeutically effective amount’ or ‘effective amount’for purposes herein is the amount determined by such considerations asare known in the art. The amount must be sufficient to enable theefficient restoration of the GI microflora thus leading to thenormalization of the function of the GI tract.

[0048] In a particular embodiment, the probiotic composition forpreventing the development of gastro-enteric infections comprises theformulation of the invention made from distilled water and volatilefractions of alfalfa, soy beans, beet, dill and mint as defined herein,at a volume ratio of, e.g., 5:1:5:15:1, and a therapeutically effectiveamount of a probiotic bacteria such as E. coli ATCC Deposit No. 202226(identical with DSM 12799) or any other probiotic bacteria.

[0049] A specific example for an gastro-enteric infection is that causedby Salmonella and the invention is of particular advantage in preventingor treating gastro-enteric infections caused thereby.

[0050] Further, the food additive or formulation of the invention, aloneor in combination with an effective amount of a probiotic microorganism,such as the E. coli strain ATCC Deposit No. 202226 (identical with DSM12799), may be used for treating or preventing infectious diarrhea,chronic diarrhea or diarrhea caused by antibiotic or chemotherapy.

[0051] According to a further specific embodiment of the invention, sucha probiotic composition for treating diarrhea may comprise distilledwater and volatile fractions of alfalfa, soy beans, beet, dill and mintat a volume ratio of, e.g., 5:1:5:15:1, and a therapeutically effectiveamount of the ATCC No. 202226 (DSMZ 12799).

[0052] The infectious diarrhea may be caused by numerous factors, forexample, by a microorganism selected from C. difficile, Salmonella,particularly S. Shigella, Campylobacter, E. coli, Proteus, Pseudomonas,Clostridium, enteric Staphylococcus. These are but few of many infectingagents.

[0053] Yet further, the probiotic compositions of the invention may beused for effectively restoring the GI microflora in a subject in need ofsuch treatment which leads to the normalization of the function of theGI tract. Such compositions may include, for example, distilled waterand volatile fractions of alfalfa, soy bean, beet, dill, mint, parsleyand cabbage, preferably at a ratio of volatile fractions of, e.g.,1:5:5:2:2:1, and a therapeutically effective amount of the probioticbacteria ATCC Deposit No. 202226 (DSM 12799).

[0054] Other purposes for which the probiotic compositions of theinvention, 5 comprising at least one probiotic bacteria having atherapeutic effect in, for example, the GI tract, include alleviation oflactose intolerance in subjects suffering from lactose intolerance,treatment of enterocolitis, treatment of constipation, for reduction ofcholesterol levels in the blood, for treatment of dyspeptic symptoms,and/or for stimulation of the immune system in subjects suffering froman immune system disorder, which may be an immune disorder caused byimmuno-suppressive therapy.

[0055] In a different aspect, the invention relates to a process forpreparing a volatile fraction of a plant extract, which processcomprises the steps of: (a) grinding plant derived matter to obtain aplant biomass; (b) mixing the plant biomass obtained in step (a) withwater at a weight proportion of preferably 3 parts water to 1 part ofthe plant biomass and stirring the same for at least 2 hours at ambienttemperature; (c) distilling the mixture obtained in step (b) underreduced pressure and at a bath temperature preferably not exceeding 38°C.; and (d) collecting the volatile fraction obtained from the steamdistillation, which fraction may further be diluted in a suitablebuffer.

[0056] The volatile fraction of the invention may be mixed with water togive the food/feed additive or formulation of the invention. Thefood/feed additive or formulation of the invention may also be preparedby mixing more than one plant volatile fraction obtained by the processof the invention. This mixture may be further mixed with water.

[0057] The volatile fractions may be prepared from may any suitablefruit, vegetable, leaf, stem or root of a plant. The plant can be, forexample, apple, citrus fruit, soy bean, beet, garlic, cabbage oralfalfa, or a herb such as parsley, mint or dill. It should be notedthat when appropriate, the formulation may further comprise volatilefractions from apicultural products such as honey or propolis or otherbeehive products. These volatile fractions may be prepared in the samemanner of the plant extract volatile fractions.

[0058] According to the process of the invention, the distillation stepis preferably carried out under reduced pressure of 5-10 mbar.

[0059] As indicated hereinbefore, the food/feed additive or formulationof the invention may be further combined with at least one probiotic orindustrial agent, to give the probiotic or industrial compositions ofthe invention. Thus, the process of preparation of the invention mayalso further comprise the step of suspending at least one viableprobiotic or industrial microorganism in the volatile fraction obtainedin said step (d) or in a mixture of such volatile fractions obtained asdescribed above.

[0060] According to a particular process of the invention, the probioticmicroorganism suspended may be the novel bacterium of the invention,derived from E. coli and deposited at the ATCC under Deposit No. 202226(and at DSM under deposit No. 12799).

[0061] Finally, the invention relates to the use of a volatile fraction(VF) of a plant extract in the preparation of a food/feed additive,wherein the VF is prepared by steam distillation of said plant extractor from apiculture product extract such as honey or propolis at a bathtemperature not exceeding 38° C.

[0062] An additional aspects of the invention include a process ofpreparing a composition including viable microorganisms. The process iseffected by suspending at least one microorganism in a formulationcontaining at least one volatile fraction (VF) of a plant extract. Theformulation is selected so as to sustain viability of themicroorganism(s) for at least 2 months at room temperature. Themicroorganism of choice may be probiotic or have industrialapplicability. Organisms which can be maintained in a formulation of thepresent invention include Escherichia spp., Alcaligenes spp.,Arthrobacter spp., Bifidobacterium spp., Lactobacillus spp., Lactococcusspp., Nitrosomonas spp. and Pseudomonas spp.

[0063] As shown in FIG. 1, in another aspect the present inventionprovides a dispenser 10 of microorganisms. Dispenser 10 includes areservoir 12 and a dispensing mechanism 14 connected thereto. Reservoir12 houses at least one microorganism in a formulation containing atleast one volatile fraction (VF) of a plant extract. The formulation isselected so as to sustain viability of the microorganism(s) for at least2 months at room temperature. Reservoir 12 may be refrigerated, say toabout 2-20° C., preferably to about 4° C. Dispenser 10 may be anairborne dispenser, so as to allow dispersion of pest control bioagents.The dispenser may be hand held. In any case, the dispenser may bedesigned to automatically dispense a predefined volume therefrom, e.g.,periodically. To this end, dispenser 10 may include a programmable orpreset control mechanism 16, valves 18, etc.

[0064] Thus, according to another aspect, the present invention providesa method of biocontrol of an organism which is effected by spreading inan endemic area at least one viable microorganism capable ofbiocontrolling the organism. The viable microorganism(s) are present ina formulation containing at least one volatile fraction (VF) of a plantextract selected so as to sustain viability of said at least onemicroorganism(s) for at least 2 months at room temperature.

[0065] Still according to another aspect the present invention providesa method of eliminating an oil pollution which is effected by spreadingin a polluted area at least one viable microorganism capable ofdegrading oil, such as Pseudomonas spp. The viable microorganism(s) ispresent in a formulation containing at least one volatile fraction (VF)of a plant extract selected so as to sustain viability of said at leastone microorganism for at least 2 months at room temperature.

[0066] Yet according to another aspect the present invention provides amethod of restoring organic fumes degrading microorganism population ina biofilter by dispensing onto the filter at least one viablemicroorganism, such as Pseudomonas spp., Nitrosomonas spp., orArthrobacter spp. capable of degrading organic fumes. The viablemicroorganism(s) is present in a formulation containing at least onevolatile fraction (VF) of a plant extract selected so as to sustainviability of said at least one microorganism for at least 2 months atroom temperature.

[0067] Still according to another aspect of the present invention thereis provided a method of preparing a starter for a fermentation processby growing a sufficient amount of a starter microorganism, such as,Lactococcus spp., Escherichia spp., Bifidobacterium spp., Lactobacillus,Lactococcus spp. or Pseudomonas spp. and suspending the startermicroorganism in a formulation containing at least one volatile fraction(VF) of a plant extract selected so as to sustain viability of saidstarter microorganism for at least 2 months at room temperature.

[0068] Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

[0069] Reference is now made to the following examples, which togetherwith the above descriptions, illustrate the invention in a non limitingfashion.

Example 1 Preparation of a “Volatile Fraction” of Plant Extracts orExtracts from Apiculture Products

[0070] Fresh vegetables obtained from commercial markets were thoroughlywashed by tap water, chopped and finely ground in an industrial blender.Distilled water was added to the vegetable biomass at the proportion of3 parts water to 1 part of the vegetable mass by weight and left understirring for at least 2 hours at ambient temperature.

[0071] The mixture was then transferred into the evaporating flask of arotatory evaporator and was evaporated at a reduced pressure (5-10 mbar)under such conditions that the temperature of the water bath did notexceed 38° C., and that of the condenser inlet was 2-5° C. About 1 literof the 2.5 kg of plant biomass.

[0072] This material could be preserved under refrigeration for at least12 months without loosing its properties, as is further exemplifiedhereinunder.

[0073] The same procedure may be carried out, replacing the plantmaterial with apiculture products such as honey or propolis.

[0074] Usually, each “volatile fraction” was prepared from a single kindof vegetable and used in different dilution to prepare differentmixtures for various purposes.

Example 2 Isolation and Growth of the Probiotic Organism E. Coli ATCCDeposit No. 202226

[0075] The probiotic organism E. coli (deposited at the ATCC under No.202226 on May 3, 1999 and at the DSM under No. 12799 on May 4, 1999) wasisolated from E. coli M-17 by sequential transfer of isolates initiatingfrom long term (2 months) survivors preserved in a formulationcontaining volatile fractions of plant derived material as is describedunder Example 1 above at 37° C. and selecting for isolates capable ofcompetition with pathogens as is further detailed below. The probioticorganism E. coli (deposited at the ATCC under No. 202226 on May 3, 1999and at the DSM under No. 12799 on May 4, 1999) was grown in thefollowing medium: (NH₄)2SO₄—5 g/liter, KH₂PO₄—13 g/liter, Na₂HPO₄—13g/liter, MgCl₂—3 g/liter, CaCl₂—0.3 g/liter, yeast extract—10 g/liter,Soy peptone—10 g/liter and glucose—5 g/liter. Additional nutrients(yeast extract—1 g/liter, Soy peptone—2.5 g/liter and glucose—90g/liter) were continuously added following the growth of the culture insuch a way that the glucose concentration in the fermentation broth waskept at a level of 2 g/liter. The pH of the fermentation broth was keptneutral by the continuous addition of 4N NH₄OH. Culturing was carriedout at 30° C. in a standard fermentation vessel with aeration of 0.5 vvmfor 16 hours, when the growth became confluent. This procedure resultedin 10¹⁰-10¹¹ cells/ml. The E. coli cells were harvested bycentrifugation, resuspended in saline and re-precipitated. The microbialbiomass could be kept in saline in the refrigerator for 48 hours withoutloosing viability.

Example 3 Preparation of Food/Feed Additive Formulation for theAccelerated Increase of Body Weight in Mammals and Avians

[0076] The mixture contained volatile fractions of: alfalfa—50 ml/liter,soy beans—200 ml/liter, beet—25 ml/liter and dill—100 ml/liter, preparedas described under Example 1 above. The balance was made up by thedistilled water.

Example 4 Preparation of a Food Supplement for the Normalization of theFunction of the Human GI Tract

[0077] The mixture contained volatile fractions of: alfalfa—50 ml/liter,soy beans—10 ml/liter, beet—50 ml/liter, dill—50 ml/liter, mint—20ml/liter, parsley—20 ml/liter and cabbage—10 ml/liter, prepared asdescribed under Example 1 above. The balance was made up by distilledwater. E. coli (ATCC 202226) cells (Example 2) were suspended in themixture at a cell concentration of 10⁷ cells/ml. NaCl (e.g., 4-10g/liter) may be optionally added for the improvement of taste.

Example 5 Preparation of a Feed Additive Formulation for the Prophylaxisof GI Infections in Mammals and Avians

[0078] The mixture contained volatile fractions of: alfalfa—50-ml/liter,soy beans—10 ml/liter, beet—50 ml/liter, dill—150 ml/liter and mint—10ml/liter, prepared as described under Example 1 above. The balance wasmade by distilled water. E. coli (ATCC 202226) cells (Example 2) weresuspended in the mixture at a cell concentration of 10⁷ cells/ml. NaCl(4-10 g/liter) may be optionally added for the improvement of the taste.

Example 6 Antagonistic Activity of E. coli (A TCC 202226) AgainstSalmonella Typhymirium (ATCC 14028)

[0079] Petri plates containing Modified Brilliant Green Agar, aselective growth medium for Salmonella, were inoculated with S.typhimirium. A 9 mm diameter well was made in the agar. A volume of thefood supplement (Example 4) was deposited in each well, and the plateswere incubated for 24 hours at 35° C. The same was repeated, but insteadof the food supplement, the fluid obtained by its filtration through amicrobiological filter membrane (pore size of 0.45 μm) was deposited inthe well.

[0080] Around each well containing the feed additive an inhibition zone(10-17 mm) devoid of S. typhimirium colonies was observed. No inhibitionzone was observed around the wells containing the filtrate free of theprobiotic organism.

Example 7 Antagonistic Activity of E. coli ATCC 202226 as well as of itsParent Strain E. coli M-17 Against Shigella Sps.

[0081] Cultures of S. flexneri, S. sonnei, E. coli (ATCC 202226) andM-17 were grown separately on a Nutrient Agar for 18-20 hours at 37° C.All the cultures were harvested in saline and diluted to an opticaldensity of 10 Klett units. Aliquots of the diluted cultures of Shigellaspecies (1 ml) alone or in combination with the diluted culture of E.coli (ATCC 202226) (1 ml) were seeded in ventilation-cup test tubescontaining Nutrient Broth (5 ml). The tubes were incubated for 24 hoursat 37° C. The number of colony-forming units (CFU) of the pathogens andof E. coli (ATCC 202226) was determined by plating the cultures onNutrient agar. The CFU numbers of two Shigella species in the pureculture and in mixed cultures with both probiotic E. coli species areshown in Table 1 below. TABLE 1 Growth of Shigella CFU/ml ProbioticOrganism S. flexneri S. sonnel E. coli ATCC 202226 <5 × 10⁴ <5 × 10⁴ E.coli M-17   1 × 10⁶   2 × 10⁶ Pure Shigella culture   6 × 10⁶   2 × 10⁶

Example 8 Application of the Food Supplement (Example 4) and ofColibacterin (Dry Formulation of E. coli M-1 7) in HospitalizedGastroenteritis Patients

[0082] A group of 60 patients that developed gastroenteritis followinghospitalization was randomly divided into 3 sub-groups with a similardistribution of sex, age and the severeness of gasroenteritis symptoms.All patients received normal supportive treatment, includingrehydration, vitamins, etc. The severity of condition did not requiretreatment with antibiotics. The first group of 20 patients received 10ml of the food supplement (Example 5) 3 times daily 30 minutes beforethe meals for 7 days. The second group of the same number receivedColibacterin (Colibacterinum siccum) as recommended by the producer (onedose twice a day 30 minutes before the meal) and the third groupreceived no supplement at all.

[0083] The onset of normalization (in days) of the symptoms ofgastroenteritis in all groups were recorded and are shown in Table 2below. TABLE 2 Onset of the normalization of the symptom, days FoodSymptoms supplement n Control Body 2.8 ± 0.2 2.7 ± 0.2 3.7 ± 0.2temperature Intoxication 2.5 ± 0.1 3.6 ± 0.1 4.6 ± 0.1 Abdominal pain3.3 ± 0.2 5.1 ± 0.2 6.1 ± 0.2 Diarrhea 1.8 ± 0.2 3.4 ± 0.2 4.4 ± 0.2

[0084] Colibacterin (Colibacterinum siccum) is the freeze-driedpreparation of live E. coli M-17 produced by BIOMED Ltd., Moscow,Russia, and recommended for use against diarrhea [Vidal Handbook:Pharmaceutical preparations in Russia (N. B. Nikolaeva, B. P. Alperovichand V. N. Sovinov, Eds.) AstraPharmService, 1997, Moscow, p. 275].

Example 9 Application of the Food Supplement (Example 4) in Patientswith Acute GI Infections

[0085] Patients with severe GI infections of various etiologies:salmonellosis, escherichiosis, shigellosis, staphylococcal infectionsand food intoxications of unknown etiology were included in the study.In all patients the hospitalization was indicated by an acute onset ofthe disease and appearance of acute gastroenteritis. The total of 186patients were treated with the food supplement and a similar group of102 patients received standard treatment.

[0086] The onset of normalization (in days) of the symptoms ofgastroenteritis in all groups were recorded and are shown in Table 3below. TABLE 3 Onset of normalization, days Symptoms Study group ControlFever 1.2 ± 0.3 2.7 ± 0.3 Weakness 1.6 ± 0.2 2.9 ± 0.3 Abdominal 1.5 ±0.2 2.4 ± 0.3 pain Diarrhea 2.3 ± 0.2 4.7 ± 0.4 Days in bed 4.8 ± 0.36.7 ± 0.4

[0087] In a separate study a group of 30 patients with intestinaltyphoid was treated with the food supplement. In 80% of patients thesymptoms of disease disappeared within 3 days. Only in 3 cases thesupplement treatment was stopped due to the development of more severechronic colitis.

Example 10 Applications of the Food Supplement (Example 4) in Patientswith GI Disorders Caused by Antibiotics

[0088] Patients with severe GI disorders were divided in 2 groups. GroupI contained 48 patients with peptic ulcer disease who developed GIdisorders after antibiotic treatment against H. pylori. Group IIcontained 22 patients that developed GI disorders following antibiotictreatment of pneumonia.

[0089] The food supplement (5 ml) was given 3 times a day before mealsfor 7 days. In both groups symptoms of diarrhea disappeared in 2-3 daysin all the patients. After the administration of the supplement,complete normalization of intestinal microflora was observed in 84.5% ofthe patients. It was demonstrated by a dramatic increase in lactobacilliand Bifidobacteria, reduction on the general count of E. coli, completedisappearance of the hemolytic E. coli and other pathogens such asStaphylococci, Proteus vulgaris and even Candida sps. In the remaining15.5% of the patients, a significant improvement was observed.

Example 11 Application of the Food Supplement (Example 5) in Patientswith Late Radiation Enterocolitis

[0090] The food supplement (10 ml, 3 times a day for 14 days, 30 minutesbefore the meals) was given to 24 patients with womb, colon and gastriccancer that developed enterocolitis following radiation therapy or acombination of radiation and chemotherapy.

[0091] Prior to the use of the food supplement, all patients complainedabout frequent and painful desire to defecate, liquid stool (4-12 timesa day) appearance of mucous (9 cases) and blood (3 cases) in the stool.

[0092] Two or three days after ingesting the supplement, all thepatients noted the lessening of pain and reduction in the number ofdefecations; the stool had a normal appearance. Four to five days laterthe diarrheal syndrome was gone, the appearance of blood and mucusceased. The blood analysis showed a strong improvement in bloodindicators.

[0093] In the control group of a similar size the symptoms persisted.

Example 12 Application of the Food Supplement (Example 4) in AIDSPatients

[0094] Patients suffering from AIDS frequently develop chronic diarrhea.A group of such patients was given 10 ml of the food supplement (Example5) 3 times a day 30 minutes before the meals for 20 days. The controlgroup received no supplement. The results of the treatment are shown inTable 4 below. TABLE 4 Parameter Food supplement Control No. of patients30 20 Average age, years 38 ± 1  36 ± 2  Daily defecation frequency: atthe onset of the Exp. 3.4 ± 0.3 3.6 ± 0.3 at the end of the Exp. 1.1 ±0.1 3.2 ± 0.3 one month after the Exp. 1.5 ± 0.2 3.7 ± 0.3 Av. onset ofnormalization 6.0 ± 0.7 remained abnormal

[0095] Patients receiving the food supplement showed normalization ofintestinal microflora: reduction in general number of coliforms,disappearance of the hemolytic E. coli, increase in the numbers ofLactobacilli and Bifidobacteria, reduction in Candida sps.

Example 13 Application of the Food/Feed Additive (Example 3) forAccelerated Weight Increase in Healthy Piglets

[0096] Healthy piglets were administrated 3 ml per os of the food/feedadditive per piglet per day until weaning. The group receiving the feedadditive gained weight at weaning on the average 1.0 kg per piglet morethan the control group.

Example 14 Application of the Feed Additive (Example 5) in HealthyPiglets

[0097] Several hundreds healthy piglets were administrated 3 ml per osof the feed additive per piglet on the first and third day afterdelivery and at weaning. Mortality was cut down by 50% compared with thecontrol group receiving standard prophylactic treatment withantibiotics. The group receiving the feed additive gained weight atweaning on the average 0.39 kg per piglet more than the control group.When the feed additive and antibiotic treatment were compared in thesame litter, the weight gain in the piglets obtaining the feed additivewas found higher by 2.4 kg than in the control.

Example 15 Application of the Feed Additive (Example 4) in PigletsShowing Diarrhea

[0098] Several hundred piglets showing diarrhea were given daily 5 mlper os of the feed additive per piglet. The control group of the samesize was treated with antibiotics: advocin, gentiamycin, amoxicillin.The symptoms of diarrhea in the group receiving the feed additivedisappeared within 1-2 days. No mortality was observed, and pigletsdeveloped normally. Antibiotics stopped diarrhea in the great majorityof piglets but the piglets remained stunted in their development.

[0099] About 70 retarded piglets, that received antibiotic treatmentagainst diarrhea for a week, and generally considered lost, were giventhe feed additive for three days. All but two survived.

Example 16 Application of the Feed Additive (Example 4) in HealthyCalves

[0100] Day-old healthy calves were administrated 5 ml of the feedadditive a day in milk during 7 days. More than 95% of the calves didnot developed diarrhea until they were 14 days old, when a few casespositive for Rotavirus were diagnosed. Normally, the incidence ofdiarrhea in this farm is 20-30%.

Example 17 Application of the Feed Additive (Example 6) in CalvesShowing Diarrhea

[0101] Calves developing diarrhea were given daily 10 ml of the feedadditive per animal with milk during 3-5 days. The symptoms of diarrheadisappeared within 1-2 days in 90-95% of calves. In the remaining 5-10%the diarrhea was caused by virus. These calves were treated withantibiotics with poor results.

Example 18 Application of the Feed Additive (Example 6) in Healthy Lambsand Goat Kids

[0102] Day-old healthy lambs and goat kids were administrated 3 ml ofthe feed additive a day in milk during 7 days. In some cases slight tomoderate signs of diarrhea were observed. These signs usuallydisappeared spontaneously or were successfully treated with an increaseddose of the feed additive (5 ml). Normally the insidence of diarrhea inthese farms is 50% for baby lambs and for goat kids.

Example 19 Application of the Feed Additive (Example 5) in Lambs andGoat Kids showing Diarrhea

[0103] The feed additive was tested in a herd suffering from pathogenicE. coli infections. In the year preceding the experiment, about 90 from120 lambs and goat kids died from diarrhea. Treatment with antibioticswas ineffective, since the disease developed suddenly and with fastmortality. Lambs and goat kids developing diarrhea were given daily 5 mlof the feed additive per animal with milk during 3-5 days. A matchinggroup of animals was treated, as normally recommended, with antibiotics.The symptoms of diarrhea disappeared within 1-2 days in about 90% of thelambs and goat kids receiving the feed additive. Their furtherdevelopment appeared normal. The control group receiving antibiotics(gentamycin) showed much poorer results. Diarrhea in this grouppersisted, calling for repeated treatment with antibiotics. Thedevelopment of the control group was severely retarded.

Example 20 Application of the Feed Additive (Example 5) in Poultry

[0104] The feed additive was added to the drinking water with theaverage uptake of 0.01 ml of the additive per day per chick during thebreeding period (42-49 days). An increase of 3.2% in weight gain,accompanied by 4% improvement in food conversion was noted in controlledtrials in broilers.

[0105] Excellent results were also obtained with turkeys of age 1 day to6 weeks. Each bird received 0.01 ml of the additive per day. A weightgain of over 10 % was observed, along with reduced mortality rate. Birdswhich still exhibited diarrhea, were treated with 0.1 ml per day of thefood additive, without any treatment by antibiotics, and showed betterrecovery from the control birds which were treated with onlyantibiotics.

Example 21 Application of the Feed Additive (Example 5) in Dogs and Cats

[0106] Application of the feed additives in puppies resulted incessation of the symptoms of diarrhea within 24-48 hours.

Example 22 Application of the Feed Additive (Example 5) in Mice;Evidence for Immunostimulation

[0107] Two-weeks-old C571B1 mice were divided into 4 groups of 10animals in each. Mice were deprived water from 4:00 pm to 8:00 am andthen given 15 ml of either water (groups 1 & 3) or the feed additive(groups 2 & 4). This treatment was repeated for 2 weeks. At the end ofthis treatment mice from groups 3 and 4 were subjected to laparotomyunder ether narcosis. A cut of 1.5 cm was made in the abdominal wall.Irritation of intestine was carried out by a dry gause. All the micewere sacrificed 24 hours after laparatomy; spleens were excised.Splenocytes were isolated and cultured on RPMI medium for 48-72 hours inthe presence of phytohemagglutinin (PHA). In the group treated with thefeed additive the proliferative activity of splenocytes reduced bylaparotomy was recovered. 1. Control group/water 3060 ± 290 2. Controlgroup/water/laparotomy 2120 ± 300 3. Test group/BioCocktail 2740 ± 3704. Test group/BioCocktail/laparotomy 3040 ± 520

Example 23 Application of the Food Additive (Example 4) in Human;Evidence for Immunostimulation

[0108] A number of patients were operated for malignant tumors of womb,cervix, stomach, and intestine. The gynecological patients (20 patients)received follow-up radiotherapy; the abdominal patients (30 patients)received chemotherapy. About half of the patients received the foodadditive twice daily for the duration of therapy. In all patientsreceiving the food additive there was observed a significantimmunostimulation, as expressed in: Gyncelogical tumors Abdominal tumorsFood Food Control Additive Control Additive T- 0.03 ± 0.01 0.10 ± 0.031.0 ± 0.1 1.6 ± 0.2 lymphocytes (CD3+, HLA−, DR+) Lymphocyte blasttrans- formation a. spon- 530 ± 110 880 ± 80  700 ± 120 1070 ± 100  taneous b. PHA- 1600 ± 360  5720 ± 2100 4400 ± 1340 13370 ± 3720  induced Phagocytes 60.7 ± 2.9  76.5 ± 2.2  57.7 ± 1.5  74.2 ± 1.9  No.(%)

Example 24 Bacteria Preservation Effect of the Formulation

[0109]E. coli cells were dispersed in a formulation prepared asdescribed under Example 1 above and was maintained for 6 months at roomtemperature. Both at the beginning and at the end of the 6 monthsperiod, samples derived from the dispersion were seeded at appropriateconsecutive dilutions on nutrient broth agar plates. The number ofcolony forming units was determined for both samples. The concentrationdetermined was 1×10⁷ in both cases, showing the effect of theformulation in preserving microorganisms. The above experiment wasperformed in parallel with Pseudomonas putida, yielding the sameresults.

[0110] Although the invention has been described in conjunction withspecific embodiments thereof, it is evident that many alternatives,modifications and variations will be apparent to those skilled in theart. Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

What is claimed is:
 1. Escherichia coli strain BU-230-98 ATCC DepositNo. 20226 (DSM 12799).
 2. A probiotic composition comprising viableEscherichia coli strain BU-230-98 ATCC Deposit No. 20226 (DSM 12799) anda formulation for maintaining viability of said Escherichia coli strain.3. The probiotic composition of claim 2, further comprising at least oneflavouring agent.
 4. The probiotic composition of claim 2, wherein saidformulation includes at least one volatile fraction (VF) of a plantextract.
 5. The probiotic composition of claim 4, wherein said volatilefraction is prepared by steam distillation of said plant extract underreduced pressure and at a bath temperature not exceeding 38° C.
 6. Theprobiotic composition of claim 4, wherein said plant extract is obtainedfrom a plant organ selected from the group consisting of leaves, stems,roots and fruit.
 7. The probiotic composition of claim 4, wherein saidplant extract is obtained from a plant selected from the groupconsisting of a vegetable and a herb.
 8. The probiotic composition ofclaim 7, wherein said vegetable is selected from the group consisting ofsoy bean, alfalfa, garlic, beet and cabbage.
 9. The probioticcomposition of claim 7, wherein said herb is selected from the groupconsisting of parsley, mint and dill.
 10. The probiotic composition ofclaim 4, further comprising a beehive product.
 11. The probioticcomposition of claim 10, wherein said beehive product is propolis. 12.The probiotic composition of claim 2, identified for preventing ortreating gastro-enteric infections or disorders.
 13. The probioticcomposition of claim 2, identified for maintaining or reinstating normalgastro-intestinal microflora.
 14. The probiotic composition of claim 2,identified for preventing or treating diarrhea.
 15. The probioticcomposition of claim 14, identified for the prevention or treatment ofgastro-enteric infection caused by an enteric pathogen.
 16. Theprobiotic composition of claim 15, wherein said pathogen is a Gramnegative bacterium or Gram positive bacterium.
 17. The probioticcomposition of claim 2, comprising distilled water and volatilefractions of beet, dill and parsley.
 18. The probiotic composition ofclaim 2, comprising distilled water and volatile fractions of beet, dilland grapefruit.
 19. The probiotic composition of claim 2, identified forpreventing or treating gastro-enteric Salmonella infection.
 20. Theprobiotic composition of claim 2, identified for preventing or treatinginfectious diarrhea, chronic diarrhea or diarrhea resulting fromantibiotic therapy, radiotherapy or chemotherapy.
 21. The probioticcomposition of claim 2, identified for treating infectious diarrhea,comprising distilled water and volatile fractions of beet, dill andparsley.
 22. The probiotic composition of claim 21, wherein saidinfectious diarrhea is caused by C. difficile, Salmonella, particularlyS. Shigella, Campylobacter, E. coli, Proteus, Pseudomonas orClostridium.
 23. The composition of claim 2, identified for normalizingthe physiological activity of the gastrointestinal tract.
 24. Aformulation comprising at least one volatile fraction (VF) of a plantextract, said volatile fraction being prepared by steam distillation ofsaid plant extract under reduced pressure and at a bath temperature notexceeding 38° C.
 25. The formulation of claim 24, wherein said plantextract is obtained from a plant organ selected from the groupconsisting of leaves, stems, roots and fruit.
 26. The formulation ofclaim 24, wherein said plant extract is obtained from a plant selectedfrom the group consisting of a vegetable and a herb.
 27. The formulationof claim 26, wherein said vegetable is selected from the groupconsisting of soy bean, alfalfa, garlic, beet and cabbage.
 28. Theformulation of claim 26, wherein said herb is selected from the groupconsisting of parsley, mint and dill.
 29. The formulation of claim 24,further comprising a beehive product.
 30. The formulation of claim 29,wherein said beehive product is propolis.
 31. A veterinary feed additivefor enhancing animal weight gain comprising at least one volatilefraction (VF) of a plant extract, said volatile fraction being preparedby steam distillation of said plant extract under reduced pressure andat a bath temperature not exceeding 38° C.
 32. The veterinary feedadditive of claim 31, comprising distilled water and volatile fractionsof beet, dill and grapefruit.
 33. A food additive for enhancing weightgain in a recipient thereof, the food additive comprising at least onevolatile fraction (VF) of a plant extract, said volatile fraction beingprepared by steam distillation of said plant extract under reducedpressure and at a bath temperature not exceeding 38° C.
 34. The foodadditive of claim 33, comprising distilled water and volatile fractionsof beet, dill and parsley.
 35. A probiotic composition comprising: (a)an effective amount of at least one viable probiotic microorganismhaving a beneficial biological or therapeutic activity in thegastrointestinal tract; and (b) at least one volatile fraction (VF) of aplant extract.
 36. The probiotic composition of claim 35, furthercomprising at least one flavouring agent.
 37. The probiotic compositionof claim 35, wherein said volatile fraction being prepared by steamdistillation of said plant extract under reduced pressure and at a bathtemperature not exceeding 38° C.
 38. The probiotic composition of claim35, wherein said probiotic microorganism is E. coli.
 39. The probioticcomposition of claim 37, wherein said probiotic microorganism is the E.coli strain BU-230-98, ATCC Deposit No.
 202226. 40. The probioticcomposition of claim 35, identified for preventing or treatinggastro-enteric infections or disorders.
 41. The probiotic composition ofclaim 35, identified for maintaining or reinstating normalgastrointestinal microflora.
 42. The probiotic composition of claim 35,identified for preventing or treating diarrhea.
 43. The probioticcomposition of claim 35, identified for the prevention or treatment ofgastro-enteric infection caused by an enteric pathogen.
 44. Theprobiotic composition of claim 43, wherein said pathogen is a Gramnegative bacterium or Gram positive bacterium.
 45. The probioticcomposition of claim 43, for treating dyspeptic symptoms.
 46. Theprobiotic composition of claim 43, for enhancing the immune response ina patient suffering from an immune disorder.
 47. The probioticcomposition of claim 46, wherein said immune disorder results fromimmune-response suppression therapy.
 48. The probiotic composition ofclaim 35, comprising distilled water and volatile fractions of beet anddill.
 49. The probiotic composition of claim 35, identified forpreventing or treating gastro-enteric Salmonella infection.
 50. Theprobiotic composition of claim 35, identified for preventing or treatinginfectious diarrhea, chronic diarrhea or diarrhea resulting fromantibiotic therapy, radiotherapy or chemotherapy.
 51. The probioticcomposition of claim 35, identified for treating infectious diarrhea,comprising distilled water and volatile fractions of beet and dill. 52.The probiotic composition of claim 51, wherein said infectious diarrheais caused by C. difficile, Salmonella, particularly S. Shigella,Campylobacter, E. coli, Proteus, Pseudomonas or Clostridium.
 53. Thecomposition of claim 35, identified for normalizing the physiologicalactivity of the gastrointestinal tract.
 54. The probiotic composition ofclaim 53, comprising distilled water and wherein said volatile fractionsare of alfalfa, soy beans, beet, dill, mint, parsley and cabbage andsaid probiotic microorganism is E. coli strain BU-230-98, ATTC DepositNo.
 202226. 55. A composition comprising: (a) at least onemicroorganism; and (b) at least one volatile fraction (VF) of a plantextract, said at least one volatile fraction is selected so as tosustain viability of said at least one microorganism for at least 2months at room temperature.
 56. The composition of claim 55, whereinsaid at least one microorganism is selected from the group consisting ofEscherichia spp., Alcaligenes spp., Arthrobacter spp., Bifidobacteriumspp., Lactobacillus spp., Lactococcus spp., Nitrosomonas spp. andPseudomonas spp.
 57. A process of preparing a volatile fraction of aplant, comprising the steps of: (a) grinding a plant derived material togive a plant biomass; (b) mixing said plant biomass with water andstirring at ambient temperature; (c) steam distilling the mixtureobtained in step (b) under reduced pressure and at a bath temperaturenot exceeding 38° C.; and (d) collecting volatile fraction obtained fromsaid steam distillation.
 58. The process of claim 57, wherein said plantextract is obtained from the leaves, stems, roots or fruit of a plant.59. The process of claim 58, wherein said plant is a vegetable or herb.60. The process of claim 59, wherein said vegetable or herb is selectedfrom the group consisting of soy, alfalfa, garlic, beet, dill, mintparsley, grapefruit and cabbage.
 61. The process of claim 57, whereinsaid reduced pressure is of 5-10 mbar.
 62. A process of preparing atherapeutic formulation, comprising the step of mixing at least onevolatile fraction (VF) of a plant extract with physiologically orveterinary acceptable additives, carriers or diluents.
 63. The processof claim 62, wherein said volatile fraction is prepared by steamdistillation of said plant extract under reduced pressure and at a bathtemperature not exceeding 38° C.
 64. A process of growing Escherichiacoli strain BU-230-98 ATCC Deposit No. 20226 (DSM 12799), the processcomprising the step of seeding a starter cloture of Escherichia colistrain BU-230-98 ATCC Deposit No. 20226 (DSM 12799) into a growth mediaand growing said Escherichia coli strain up to optical density of 15-30at 650 nm.
 65. A process of manufacturing a probiotic composition, theprocess comprising the step of suspending viable Escherichia coli strainBU-230-98 ATCC Deposit No. 20226 (DSM 12799) in a formulation formaintaining viability of said Escherichia coli strain.
 66. The processof claim 65, wherein said formulation includes at least one volatilefraction (VF) of a plant extract.
 67. The process of claim 66, whereinsaid volatile fraction is prepared by steam distillation of said plantextract under reduced pressure and at a bath temperature not exceeding38° C.
 68. The process of claim 66, wherein said plant extract isobtained from a plant organ selected from the group consisting ofleaves, stems, roots and fruit.
 69. The process of claim 66, whereinsaid plant extract is obtained from a plant selected from the groupconsisting of a vegetable and a herb.
 70. The process of claim 69,wherein said vegetable or herb is selected from the group consisting ofsoy bean, parsley, mint, dill, alfalfa, garlic, beet and cabbage. 71.The process of claim 66, further comprising the addition of a beehiveproduct.
 72. The process of claim 71, wherein said beehive product ispropolis.
 73. The process of claim 65, wherein said at least onevolatile fraction is of alfalfa, soy beans, beet, dill and mint.
 74. Aprocess of preparing a probiotic composition, the process comprising thestep of suspending an effective amount of at least one viable probioticmicroorganism having a beneficial biological or therapeutic activity inthe gastrointestinal tract in an formulation containing at least onevolatile fraction (VF) of a plant extract.
 75. The process of claim 74,wherein said volatile fraction is prepared by steam distillation of saidplant extract under reduced pressure and at a bath temperature notexceeding 38° C.
 76. The process of claim 74, wherein said probioticmicroorganism is E. coli.
 77. The process of claim 75, wherein saidprobiotic microorganism is the E. coli strain BU-230-98, ATCC DepositNo.
 202226. 78. The process of claim 74, wherein said at least onevolatile fraction includes volatile fractions of alfalfa, soy beans,beet, dill, mint, parsley and cabbage.
 79. A process of preparing acomposition including viable microorganisms, the process comprising thestep of suspending at least one microorganism in a formulationcontaining at least one volatile fraction (VF) of a plant extract, saidat least one volatile fraction is selected so as to sustain viability ofsaid at least one microorganism for at least 2 months at roomtemperature.
 80. The process of claim 79, wherein said at least onemicroorganism is selected from the group consisting of Esherichia spp.,Alcaligenes spp., Arthrobacter spp., Bifidobacterium spp., Lactobacillusspp., Lactococcus spp., Nitrosomonas spp. and Pseudomonas spp.
 81. Adispenser of microorganisms comprising a reservoir and a dispensingmechanism being connected thereto, said reservoir housing at least onemicroorganism in a formulation containing at least one volatile fraction(VF) of a plant extract, said at least one volatile fraction is selectedso as to sustain viability of said at least one microorganism for atleast 2 months at room temperature.
 82. The dispenser of claim 81,wherein said dispenser is airborne.
 83. The dispenser of claim 81,wherein said dispenser is hand held.
 84. The dispenser of claim 81,wherein said dispenser is designed to automatically dispense apredefined volume.
 85. The dispenser of claim 81, wherein said dispenseris designed to automatically dispense said predefined volumeperiodically.
 86. A method of biocontrol of an organism, the methodcomprising the step of spreading in an endemic area at least one viablemicroorganism capable of biocontrolling the organism, said at least oneviable microorganism being in a formulation containing at least onevolatile fraction (VF) of a plant extract, said at least one volatilefraction is selected so as to sustain viability of said at least onemicroorganism for at least 2 months at room temperature.
 87. A method ofeliminating an oil pollution, the method comprising the step ofspreading in a polluted area at least one viable microorganism capableof degrading oil, said at least one viable microorganism being in aformulation containing at least one volatile fraction (VF) of a plantextract, said at least one volatile fraction is selected so as tosustain viability of said at least one microorganism for at least 2months at room temperature.
 88. A method of restoring organic fumesdegrading microorganism population in a biofilter, the method comprisingthe step of dispensing onto the filter at least one viable microorganismcapable of degrading organic fumes, said at least one viablemicroorganism being in a formulation containing at least one volatilefraction (VF) of a plant extract, said at least one volatile fraction isselected so as to sustain viability of said at least one microorganismfor at least 2 months at room temperature.
 89. A food productsupplemented with viable Escherichia coli strain BU-230-98 ATCC DepositNo. 20226 (DSM 12799).
 90. The food product of claim 89, comprising atleast one milk derived substance.
 91. The food product of claim 90,selected from the group consisting of a cheese and a yogurt.
 92. Amethod of preparing a starter for a fermentation process, the methodcomprising the step of growing a sufficient amount of a startermicroorganism and suspending said starter microorganism in a formulationcontaining at least one volatile fraction (VF) of a plant extract, saidat least one volatile fraction is selected so as to sustain viability ofsaid starter microorganism for at least 2 months at room temperature.